US12163125B2ActiveUtilityA1

Engineered class 2 type V CRISPR systems

91
Assignee: SCRIBE THERAPEUTICS INCPriority: Dec 3, 2020Filed: Jan 10, 2022Granted: Dec 10, 2024
Est. expiryDec 3, 2040(~14.4 yrs left)· nominal 20-yr term from priority
Inventors:Gayathri VijayakumarSean HigginsIsabel ColinSarah DennyBrett T. StaahlBenjamin OakesAngus SidoreSuraj Makhija
C12N 2750/14143C12N 2320/50C12N 15/907C12N 15/86C12N 15/111C12N 15/11C12N 2800/80C12N 15/85C12N 15/113C12N 9/22C12N 2310/20C12N 15/102
91
PatentIndex Score
5
Cited by
167
References
28
Claims

Abstract

Provided herein are engineered Class 2, Type V nucleases and guide RNAs useful for the editing of target nucleic acids. Also provided are methods of making and using such variants to modify nucleic acids.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
       1. A chimeric Class 2, Type V CRISPR protein comprising:
 a. a non-target strand binding (NTSB) domain obtained from Deltaproteobacteria comprising the sequence of SEQ ID NO: 2335, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto; 
 b. a helical I-II domain obtained from Deltaproteobacteria comprising the sequence of SEQ ID NO: 2336, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto; and 
 c. a RuvC-I domain obtained from Planctomycetes comprising the sequence of SEQ ID NO: 2352, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto, wherein the RuvC-I domain comprises a proline amino acid residue at position 793 relative to SEQ ID NO: 336, and wherein the chimeric Class 2, Type V CRISPR protein exhibits improved specificity relative to SEQ ID NO: 336. 
 
     
     
       2. The chimeric Class 2, Type V CRISPR protein of  claim 1 , comprising a helical I-I domain, a helical II domain, an oligonucleotide binding domain (OBD)-I domain, an OBD-II, a target strand loading (TSL) domain, and a RuvC-II domain. 
     
     
       3. The chimeric Class 2, Type V CRISPR protein of  claim 2 , wherein the helical I-I domain, helical II domain, OBD-I domain, OBD-II, TSL domain, and RuvC-II domain are each obtained from Planctomycetes. 
     
     
       4. The chimeric Class 2, Type V CRISPR protein of  claim 3 , wherein the helical I-I domain obtained from Planctomycetes comprises the sequence of SEQ ID NO: 2343, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. 
     
     
       5. The chimeric Class 2, Type V CRISPR protein of  claim 3 , wherein the helical II domain obtained from Planctomycetes comprises the sequence of SEQ ID NO: 2351, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto. 
     
     
       6. The chimeric Class 2, Type V CRISPR protein of  claim 3 , wherein the OBD-I domain obtained from Planctomycetes comprises the sequence of SEQ ID NO: 2342, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. 
     
     
       7. The chimeric Class 2, Type V CRISPR protein of  claim 3 , wherein the OBD-II domain obtained from Planctomycetes comprises the sequence of SEQ ID NO: 2347, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. 
     
     
       8. The chimeric Class 2, Type V CRISPR protein of  claim 3 , wherein the TSL domain obtained from Planctomycetes comprises the sequence of SEQ ID NO: 2349, or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. 
     
     
       9. The chimeric Class 2, Type V CRISPR protein of  claim 3 , wherein the RuvC-II domain obtained from Planctomycetes comprises the sequence of SEQ ID NO: 2350, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. 
     
     
       10. The chimeric Class 2, Type V CRISPR protein of  claim 1 , wherein the RuvC-I domain comprises a substitution at amino acid position A708 relative to the Planctomycetes sequence of SEQ ID NO: 2. 
     
     
       11. The chimeric Class 2, Type V CRISPR of  claim 10 , wherein the substitution is A708K. 
     
     
       12. The chimeric Class 2, Type V CRISPR protein of  claim 1 , wherein the RuvC-I domain comprises a substitution at amino acid position I658 relative to the Planctomycetes sequence of SEQ ID NO: 2. 
     
     
       13. The chimeric Class 2, Type V CRISPR of  claim 1 , wherein the substitution is I658V. 
     
     
       14. The chimeric Class 2, Type V CRISPR protein of  claim 1 , comprising the sequence of SEQ ID NO: 416 or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. 
     
     
       15. The chimeric Class 2, Type V CRISPR protein of  claim 1 , comprising the sequence of SEQ ID NO: 416. 
     
     
       16. The chimeric Class 2, Type V CRISPR protein of  claim 1 , comprising a substitution of G223S relative to the sequence of SEQ ID NO: 416. 
     
     
       17. The chimeric Class 2, Type V CRISPR protein of  claim 16 , comprising an insertion of an arginine amino acid (R) at position 26 relative to the sequence of SEQ ID NO: 416. 
     
     
       18. The chimeric Class 2, Type V CRISPR protein of  claim 17 , comprising a substitution of L169K relative to the sequence of SEQ ID NO: 416. 
     
     
       19. The chimeric Class 2, Type V CRISPR protein of  claim 16 , comprising a substitution of L169K relative to the sequence of SEQ ID NO: 416. 
     
     
       20. The chimeric Class 2, Type V CRISPR protein of  claim 1 , comprising a substitution of L169K and a substitution of G223S relative to the sequence of SEQ ID NO: 416. 
     
     
       21. The chimeric Class 2, Type V CRISPR protein of  claim 1 , comprising an insertion of R at position 26 and a substitution of G223S relative to the sequence of SEQ ID NO: 416. 
     
     
       22. The chimeric Class 2, Type V CRISPR protein of  claim 1 , comprising an insertion of R at position 26, a substitution of G223S, and a substitution of L169K relative to the sequence of SEQ ID NO: 416. 
     
     
       23. The chimeric Class 2, Type V CRISPR protein of  claim 1 , comprising one or more improved characteristics compared to the reference protein of SEQ ID NO: 2. 
     
     
       24. The chimeric Class 2, Type V CRISPR protein of  claim 23 , wherein the one or more improved characteristics comprise improved stability of a ribonuclear protein (RNP) complex comprising the Class 2, Type V CRISPR protein and a guide ribonucleic acid (gRNA), increased binding affinity between the Class 2, Type V CRISPR protein and gRNA, improved kinetics of RNP complex formation, higher percentage of cleavage-competent RNP, increased RNP binding affinity to a target nucleic acid, improved unwinding of the target nucleic acid, increased editing activity, increased editing efficiency, increased editing specificity for the target nucleic acid, decreased off-target editing or cleavage, increased nuclease activity of the Class 2, Type V CRISPR protein, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, increased binding of the non-target strand of DNA, or increased resistance to nuclease activity. 
     
     
       25. A chimeric Class 2, Type V CRISPR protein comprising the sequence of SEQ ID NO: 416, or a sequence having at least 90% identity thereto, wherein the Class 2, Type V CRISPR protein comprises a proline amino acid at position 793 relative to SEQ ID NO: 336, wherein the chimeric Class 2, Type V CRISPR protein comprises domains from two different Class 2, Type V CRISPR proteins, and wherein the chimeric Class 2, Type V CRISPR protein exhibits improved specificity relative to SEQ ID NO: 336. 
     
     
       26. The chimeric Class 2, Type V CRISPR protein of  claim 25 , comprising:
 a. an NTSB domain obtained from Deltaproteobacteria comprising the sequence of SEQ ID NO: 2335, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto; 
 b. a helical 1-II domain obtained from Deltaproteobacteria comprising the sequence of SEQ ID NO: 2336, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto; and 
 c. a RuvC-I domain obtained from Planctomycetes comprising the sequence of SEQ ID NO: 2352, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto. 
 
     
     
       27. The chimeric Class 2, Type V CRISPR protein of  claim 25 , comprising one or more improved characteristics compared to the reference protein of SEQ ID NO: 2. 
     
     
       28. A chimeric Class 2, Type V CRISPR protein comprising the sequence of SEQ ID NO: 416.

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